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Biosurfactant biosynthesis by Alcanivorax borkumensis and its role in oil biodegradation

Nature Chemical Biology volume 21, pages 1631–1641 (2025)Cite this article

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Abstract

The marine bacterium Alcanivorax borkumensis degrades alkanes derived from phytoplankton, natural hydrocarbon seeps and oil spills. We study the biosynthesis and function of a glycine-glucolipid biosurfactant from A. borkumensis for alkane degradation and identify a gene cluster encoding a nonribosomal peptide synthetase, glycosyltransferase and phosphopantetheinyl transferase. Analyses of A. borkumensis mutants and expression studies reveal that the nonribosomal peptide synthetase catalyzes the synthesis of the aglycone (tetra-d-3-hydroxydecanoyl-glycine) from glycine and d-3-hydroxydecanoyl-CoA, to which a glucose moiety is added by the glycosyltransferase. Deficiency in glycine-glucolipid impairs the ability of mutant cells to attach to the oil–water interface, compromises growth on hexadecane and affects carbon storage. The glycine-glucolipid is essential for biofilm formation on oil droplets and uptake of alkanes. The high incidence of Alcanivorax at oil-polluted sites can in part be explained by the accumulation of the glycine-glucolipid on the cell surface, effectively making the cells themselves act as biosurfactants.

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Fig. 1: gglsA encodes an NRPS involved in glycine-glucolipid synthesis.
Fig. 2: Phylogenetic tree of C domains of NRPS proteins.
Fig. 3: gglsB encodes a glycosyltransferase essential for glycine-glucolipid synthesis.
Fig. 4: Growth of A. borkumensis WT and ΔgglsA and ΔgglsB cells on pyruvate and hexadecane.
Fig. 5: Growth and attachment to the oil–water interface of A. borkumensis mutants deficient in the glycine-glucolipid.
Fig. 6: Ultrastructure of WT A. borkumensis and ΔgglsA and ΔgglsB mutant cells deficient in glycine-glucolipid formation.

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Data availability

The data generated in this study are provided within the article and in Supplementary Information. The complete genome sequence of A. borkumensis SK2, including the gene cluster ABO_1784, ABO_1783 and ABO_1782, can be retrieved at www.ncbi.nlm.nih.gov/nuccore/AM286690.1. The following databases were used in this study: Carbohydrate Active enZYmes (www.cazy.org) and PKS/NRPS Analysis website (https://nrps.igs.umaryland.edu/). Data are also available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank the Microscopy Core Facility of the Medical Faculty at the University of Bonn for providing support and instrumentation funded by the German Research Foundation (grant 388171357). We thank L. Sundermeyer for support with the initial isolation of the gglsA–gglsB–gglsC gene cluster and F. Diaz and L.-M. Kirschen for their experimental support. This work was supported by the German Ministry of Education and Research (BioProMare, GlycoX; grant 161B0866A to K.-E.J., S.T. and S.K.; 161B0866B to L.M.B. and T.K.; 161B0866C to P.D.) and by the German Research Foundation (grant EXC-2070–390732324, PhenoRob to P.D.; EXC-2186, Fuel Science Center FSC to L.M.B.).

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Authors and Affiliations

  1. Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, Bonn, Germany

    Jiaxin Cui, Maximilian Fassl, Vaisnavi Vasanthakumaran, Maya Marita Dierig, Georg Hölzl & Peter Dörmann

  2. Institute of Applied Microbiology-iAMB, RWTH Aachen University, Aachen, Germany

    Tobias Karmainski & Till Tiso

  3. Institute for Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Jülich, Germany

    Sonja Kubicki, Lars M. Blank & Karl-Erich Jaeger

  4. Institute of Bio- and Geosciences IBG 1, Biotechnology, Forschungszentrum Jülich, Jülich, Germany

    Stephan Thies & Karl-Erich Jaeger

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Contributions

K.-E.J., S.T., L.M.B. and P.D. conceived the study. J.C., M.F., V.V., M.M.D., G.H., T.K., T.T., S.K. and S.T. designed and performed most experiments. S.T., K.-E.J., L.M.B. and P.D. acquired funding and supervised the project. K.-E.J., S.T., S.K., L.M.B., T.T., T.K. and P.D. wrote the paper with input from all authors.

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Extended data

Extended Data Fig. 1 The protein sequence of the nonribosomal peptide synthetase (NRPS) GglsA (ABO_1784).

The domains of the NRPS GglsA are depicted in different colors (C domain, yellow; A domain, bright blue; T domain, golden; TE domain, pink). The two serines, Ser1039 (attachment site of phosphopantetheine group) and Ser1177 (active site of TE domain) are shown in bold red.

Extended Data Fig. 2 GglsA containing the A domain of E. coli EntF catalyzes the synthesis of the serine-glucolipid.

a, The GglsA protein carrying the A domain of the E. coli EntF protein (GglsA-EntFA) was expressed in E. coli, and lipids measured by LC-MS/MS. The MS/MS spectrum displays the parental mass at 803.5788 m/z indicative for the aglycone H(O-10:0)4Ser which differs from the aglycone of the glycine-glucolipid H(O-10:0)4Gly (773.5905 m/z) (Fig. 1c) by 29.9883 m/z equivalent to one HC-OH unit (difference between Ser/Gly). The same mass difference was observed between the fragment peak of H(O-10:0)4Ser (106.0436 m/z) and H(O-10:0)4Gly (76.0386 m/z). The structure of the serine-containing aglycone on the right shows the calculated masses. b, The chimeric GglsA-EntFA protein containing the A domain of E. coli EntF catalyzes the synthesis of the aglycone H(O-10:0)4Ser carrying a serine moiety instead of glycine.

Source data

Extended Data Fig. 3 Reaction mechanism of GglsA.

a, The GglsA protein carrying the Ser1177Ala mutation to disable thioesterase activity was expressed in E. coli. Glycine-glucolipid intermediates bound to the phosphopantetheine group were released with cysteamine and analyzed by LC-MS. The MS/MS spectrum of the peak eluting at 48.3 min (inset) shows the fragmentation of the proton adduct of the di-cysteamine derivative of the aglycone H(O-10:0)4Gly carrying four acyl groups. b, The GglsA construct containing the C-A-T domains but lacking the TE domain was expressed in E. coli. The aglycones produced were released by cysteamine cleavage. LC-MS analysis revealed the synthesis of four different aglycones with four, three, two or one 3-hydroxydecanoyl groups with different retention times and MS/MS spectra. The insets show the LC-MS chromatograms for the parental ions (x axis, time in min).

Source data

Extended Data Fig. 4 Nonpolar lipid accumulation in A. borkumensis ΔgglsA and ΔgglsB mutant cells.

Cells were grown in pyruvate or hexadecane medium. Lipid extracts equivalent to the same amount of total lipids (measured by gas chromatography of fatty acid methyl esters) were separated by thin-layer chromatography (TLC) and stained with iodine vapor. Storage lipids were identified by co-migration with standards or by isolation and mass spectrometry. TAG, triacylglycerol; WE, wax ester.

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Cui, J., Fassl, M., Vasanthakumaran, V. et al. Biosurfactant biosynthesis by Alcanivorax borkumensis and its role in oil biodegradation. Nat Chem Biol 21, 1631–1641 (2025). https://doi.org/10.1038/s41589-025-01908-1

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  • DOI: https://doi.org/10.1038/s41589-025-01908-1

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